Low temperature adaptor for evaporative light detection

ABSTRACT

The present invention is directed to a system for converting between single flow and split flow evaporative light scattering detection devices for detecting samples in a mobile phase. Included in the system is a evaporative light scattering detection device, a low temperature adaptor, and a connection tube for providing a fluid connection between the evaporative light scattering detection device and the low temperature adaptor.

FIELD OF THE INVENTION

Applicants' invention is directed to the field of evaporative lightscattering detection and methods.

BACKGROUND OF THE INVENTION

Evaporative light scattering detection is a method of detecting samplesthat have been previously separated in various chromatography methodssuch as, for example, High Performance Liquid Chromatography (HPLC),Gel-Permeation Chromatography (GPC), High Performance CentrifugalPartition Chromatography (HPCPC), Field Flow Fractionation (FFF), andSupercritical Fluid Chromatography (SFC). Evaporative light scatteringdetection is preferably used when the sample components (e.g.,components to be detected) have lower volatility than the mobile phase.A wide variety of sample types can be detected in evaporative lightscattering detection. Such sample types include, for example, lipids,triglycerides, surfactants, polymers, underivatized fatty and aminoacids, carbohydrates and pharmaceuticals.

Generally, evaporative light scattering detection involves four mainsteps: 1) nebulization of the chromatography effluent, (which consistsof the mobile phase and the sample), into an aerosol of particles, 2)evaporation of the mobile phase, 3) light scattering by the sampleparticles, and 4) detection of the scattered light. There are twoprincipal types of devices used in evaporative light scatteringdetection known in the art. In the first type (the “single flow”design), the nebulized chromatography effluent is immediately introducedinto a heated drift tube where the mobile phase is evaporated. Thesample particles are then flowed from the heated drift tube to anoptical cell where light scattering and detection occurs. One suchexample of this type of device (the Alltech Model 500 ELSD) is sold bythe assignee of this application, ALLTECH ASSOCIATES, INC. Detailsconcerning the design and operating parameters for such a device aredisclosed in the Operating Manual for the Alltech Model 500 ELSD, whichis incorporated herein by reference.

In the second type of device, (the “split-flow” design), the nebulizedchromatography effluent is first flowed through a nebulization chamberbefore entering the heated drift tube. In the nebulization chamber, thenebulized chromatography effluent is split, namely, the larger dropletsare eliminated by condensation/impaction on the walls of thenebulization chamber. This condensate is drained to waste. Only thesmaller nebulized droplets are subsequently flowed to the drift tubewhere the mobile phase (which is now free of the larger droplets) ismore easily evaporated. Thereafter, the sample particles are flowed tothe optical cell for light scattering and detection. Devices of thisdesign type are available from, for example, SEDERE or EUROPSEPINSTRUMENTS.

The above-described design types have particular advantages depending onthe mobile phase and the sample type. The single flow design ispreferred for use in applications involving relatively non-volatilesample types and volatile organic mobile phases. Because all of thesample enters the optical cell in this design, response and sensitivityis maximized.

However, the split-flow design is preferably used with highly aqueousmobile phases and semi-volatile sample types. Highly aqueous mobilephases generally require higher evaporation temperatures. If the sampleis volatile at these higher evaporation temperatures, sample loss isincurred during the evaporation step resulting in poorer sensitivity. Byusing the split-flow design, the evaporation temperature is reduced.This is accomplished by removing the larger mobile phase droplets in thenebulized chromatography effluent before the evaporation step. Byremoving the larger droplets, a smaller and more uniform particle sizedistribution is achieved in the mobile phase, which leads to lowerevaporation temperatures. The lower evaporation temperatures, in turn,lead to less sample loss during the evaporation step. However, fornon-volatile sample types and organic mobile phases, the split-flowdesign is generally less preferred because some of the non-volatilesample may be lost during the splitting of the chromatography effluent.

Another problem with devices of the split-flow design is that the splitratio of the sample (i.e., the amount that goes to waste versus theamount that is ultimately detected) is affected by, among other things,the laboratory temperature. In other words, fluctuations in laboratorytemperatures lead to fluctuations in droplet size in the nebulizedchromatography effluent. Thus, as ambient and/or laboratory temperaturesfluctuate, the split ratio and corresponding reproducibility of sampledetection may vary from run to run.

As is evident from the above-discussion, depending on the mobile phaseand the sample type being detected, one evaporative light scatteringdetection design and method is advantageous over the other. However,laboratories often work with both aqueous and organic mobile phases andvarious sample types with different volatilities. Ideally, laboratorieswould have available both design types for evaporative light scatteringdetection. However, in order to have this benefit, the laboratory wouldneed to purchase two separate devices, which can be expensive. It wouldbe advantageous and constitute an improvement in the art if anevaporative light scattering detection device and system were developedwhich could be quickly and inexpensively converted between the singleflow and split flow designs. Applicants have developed such a device andsystem. Moreover, with respect to the split-flow design, Applicantsinvention addresses the problem of the variation in split ratio causedby fluctuating laboratory temperatures.

SUMMARY OF THE INVENTION

In one respect, the present disclosure is directed to a system forevaporative light scattering detection which allows for quick and easyconversion between a single flow design and a split flow design,depending on the mobile phase and sample types to be detected. Thesystem includes an evaporative light scattering detection devicecomprising a removably attached nebulizer in fluid communication with aheated drift tube, a light source, and a detector for detectingscattered light. The system also includes a low temperature adaptorcomprising a nebulization chamber and a coil. The system furtherincludes a connection tube for providing a fluid connection between thelight scattering detection device and the low temperature adaptor forconverting from a single flow to the split flow designs. One end of theconnection tube is attached to the low temperature adaptor and the otherend of the connection tube is removably attached to the evaporativelight scattering detection device such that the connection tube providesfluid communication between the low temperature adaptor and theevaporative light scattering device. The low temperature adaptor isconnected to the evaporative light scattering device by first removingthe nebulizer from the detection device and attaching in its place theconnection tube to provide fluid communication between the lowtemperature adaptor and the detection device. The low temperatureadaptor further comprises a nebulizer. The nebulizer for the lowtemperature adaptor may be the nebulizer removed from the evaporativelight scattering device or a second nebulizer.

The low temperature adapter in the above system further preferablycomprises a sweep gas channel for introducing into the nebulizationchamber sweep gas independently of the nebulizing gas. The sweep gas isfor assisting in the evaporation of the mobile phase. Also, heat tape ispreferably affixed to the nebulization chamber and the coil of the lowtemperature adaptor in the above system at pre-determined intervals forcontrolling the temperature of the nebulization chamber and coil.

In another respect, the disclosure is directed to a low temperatureadaptor for a light scattering detection device which reduces thetemperature required to evaporate the mobile phase. The low temperatureadaptor is especially preferred for aqueous mobile phases andsemi-volatile sample types. The low temperature adaptor comprises anebulization chamber, a coil and a connection tube for removablyattaching the low temperature adaptor to the evaporative lightscattering detection device such that the connection tube provides afluid connection between the low temperature adaptor and the evaporativelight scattering detection device. Heat tape is preferably affixed tothe nebulization chamber and the coil at pre-determined intervals forcontrolling the temperature of the nebulization chamber and coil. Thelow temperature adaptor preferably further includes a sweep gas channelfor introducing sweep gas into the nebulization chamber independently ofnebulizing gas. Finally, the low temperature adapter further includes anebulizer. The nebulizer may be the nebulizer removed from theevaporative light scattering detection device prior to connecting thelow temperature adapter or a second nebulizer.

In another aspect, the disclosure concerns a method of evaporative lightscattering detection which is substantially resistant to fluctuations inambient temperature conditions. By substantially resistant tofluctuations in ambient temperature conditions, it is meant that thedetection device of this invention provides consistent detection whenlaboratory temperatures fluctuate of from about 15° C. to about 40° C.The method comprises flowing nebulized chromatography effluentcomprising mobile phase and sample to be detected through a nebulizationchamber, wherein the temperature of the nebulization chamber iscontrolled by a heat source; reducing the particle size distribution ofthe nebulized chromatography effluent in the nebulization chamber;evaporating the mobile phase; and detecting the sample by evaporativelight scattering detection. Preferably, the temperature in thenebulization is controlled by heat tape affixed to the nebulizationchamber at predetermined intervals.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating the principles of operationof an evaporative light scattering detection device.

FIG. 1a is an isometric view of the configuration for an evaporativelight scattering detection device.

FIG. 2 is a cross-section view along line A—A of FIG. 1 showing thedrift tube assembly.

FIG. 3 is a cross-section along lines A—A and B—B of FIG. 1 of thenebulizer and nebulizer adaptor.

FIG. 3a is a top view of the nebulizer and nebulizer adaptor shown inFIG. 3.

FIG. 3b is a side view of the nebulizer body portion of the nebulizershown in FIG. 3.

FIG. 4 is a cross-section view along line A—A of FIG. 1 showing themanner of connecting the nebulizer to the drift tube assembly.

FIG. 5 is a perspective isometric view of the configuration for a lowtemperature adaptor.

FIG. 6 is a top isometric view of the configuration for a lowtemperature adaptor.

FIG. 7 is a perspective view of the coil and nebulization chamber of thelow temperature adaptor.

FIG. 8a is a partial cross-section view along line A—A of FIG. 6 showingthe attachment of the nebulizer union to the nebulization chamber of thelow temperature adaptor.

FIG. 8b is a cross-section view along lines A—A and C—C of FIG. 6.

FIG. 8c is a top plan view of FIG. 8a.

FIG. 9 is a diagram depicting sample flow through the low temperatureadaptor and the evaporative light scattering device.

FIG. 10 is a cross-section view along line D—D of FIG. 9 showing theconnection between the low temperature adaptor, connection tube andevaporative light scattering device.

FIGS. 11-21 are chromatograms demonstrating the use of the evaporativelight scattering detection device and low temperature adaptor disclosedherein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

FIGS. 1-4 illustrate an evaporative light scattering detection device ofthe single flow design. FIG. 1 provides an overview of the principal ofoperation of an evaporative light scattering detection device 10. Thescattering device 10 has a connector 12. The connector 12 provides afluid connection between the chromatography column (not shown) and theevaporative light scattering device 10. The connector 12 is preferablymade from stainless steel and is threadingly engaged to nebulizerbracket 16. Chromatography effluent is flowed into the evaporative lightscattering device 10 through channel 13 in connector 12. Nebulizerbracket 16 removably attaches nebulizer 14 (which consists of pieces101, 102 and 103 discussed below) to drift tube assembly 18. Thenebulizer 14 contains a nebulizer needle (not shown). The drift tubeassembly 18 surrounds a central heated drift tube channel 11. Finally, alaser light source 20, a photodetector 22 and amplifier 24 are provided.

In operation, and with reference to FIG. 1, the chromatography effluentis flowed through connector 12 to nebulizer 14. The chromatographyeffluent is directed through the nebulizer needle (not shown). Uponexiting the nebulizer needle, the chromatography effluent is impacted bynebulizing gas to form an aerosol of droplets, preferably of generallyuniform size. The nebulizing gas may include any gas that is inert tothe sample such as helium, carbon dioxide, air or nitrogen, and ispreferably nitrogen.

The nebulized chromatography effluent is then flowed through channel 11in the drift tube assembly 18. In channel 11, the mobile phase isevaporated leaving behind the non-volatile sample particles. Thenon-volatile sample particles are flowed through channel 11 to the lightscattering zone 19 for detection. A light source 20 emits light, whichthe sample particles scatter. The scattered light is then detected atthe photodetector 22. The photodetector 22 then produces a signal whichis sent to an amplifier 24 though analog outputs in the photodetector.

The light source 20 is preferably a Class IIIA laser product with a 650nm laser diode, 5 mW maximum power, collimating optics, and polarized. Apreferred laser light source is available from COHERENT, as part no.VLM3-5L. The photodetector 22 is preferably made from a siliconphotodiode. A preferred photodetector 22 is available from HAMAMATSU, aspart no. S2386-8K. The photodetector 22 is preferably located at a 90degree angle from the light source 20. A light trap 31 is alsopreferably located at an 180 degree angle from the light source 20 tocollect any light not scattered by the sample particles in the aerosolstream. After detection in the detection zone, the sample particles areflowed through an exhaust line (now shown) to waste. Preferably theexhaust is flowed to a fume hood or other ventilation device locatedclose to the detector to remove the detector exhaust from thelaboratory.

A configuration and preferred flow paths for the sample and thenebulizing gas of an evaporative light scattering device 10 isillustrated by FIG. 1a. Preferably included in the evaporative lightscattering device 10 is liquid pressure sensor 33, liquid back pressurerelief valve 34, gas shut off valve 35, mass flow sensor 36 and massflow control 36 a, gas pressure sensor 37, temperature control 38 a,power input 39, logic board 38 (which includes a microprocessor) andfront control panel 38 c. The logic board 38 is in electricalcommunication (not shown) with front panel display 38 c.

The liquid back pressure relief valve 34 is in fluid communication withthe nebulizer 14 via a stainless steel flow path (not shown). If thenebulizer needle becomes blocked or chromatography effluent backpressure otherwise exceeds a pre-determined level, the back pressurerelief valve 34 diverts the flow of chromatography effluent fromnebulizer 14 to waste. A preferred back pressure relief valve is adiaphragm back pressure regulator available from the assignee of thisapplication ALLTECH ASSOCIATES, INC. The liquid pressure sensor 33 isalso in fluid communication with the nebulizer 14 via a stainless steelflow path (not shown). The liquid pressure sensor monitors thechromatography effluent back pressure in the nebulizer 14. The liquidpressure sensor 33 is also in electrical communication (not shown) withlogic board 38 and communicates pressure readings to logic board 38 fordisplay on front panel 38c. A preferred liquid pressure sensor isavailable from KELLER PSI, as part no. PR-6-10. The sample in thechromatography effluent is flowed from nebulizer 14 through drift tubeassembly 18 to detection zone 19 for sample detection.

The nebulizing gas is flowed from gas inlet 30 a to gas filter 30 whereimpurities are removed from the nebulizing gas. A preferred gas filterhas a 1 micron pore size and is available from WEBSTER ASSOC., as partno. FILNO5012YE. The nebulizing gas is then flowed from gas filter 30through, respectively, gas pressure sensor 37, gas shut-off valve 35,mass flow controller 36 a, mass flow sensor 36 to nebulizer 14. Each ofthe foregoing devices are in gas communication via TEFLON tubing. Thegas pressure sensor 37 monitors the nebulizing gas and is in electricalcommunication with logic board 38. The gas pressure sensor 37 triggersan alarm if the nebulizing gas pressure falls below or exceedspre-determined levels. A preferred gas pressure sensor is available fromIC SENSORS, as part no. 1201A-100G-3L. The gas shut-off valve 35 is inelectrical communication with logic board 38 and may be triggered tostop the flow of nebulizing gas as, for example, between detection runs.The mass flow controller 36 a is manually operated for setting the flowrate of the nebulizing gas. A preferred mass flow controller allows fornebulizing gas flowrates of from 0-5 Standard Liters Per Minute (SLPM)and is available from CONDYNE, as part no. 100-6(14)4. The mass flowsensor 36 measures the flow rate of the nebulizing gas and is inelectrical communication with logic board 38, which in turn displays thenebulizing gas flowrate on front control panel 38 c. A preferred massflow sensor is available from INTEGRATED ELECTRONICS, as part no.AWM43600V.

The logic board 38 further processes the analog signal from thephotodetector 22 and contains a microprocessor for displaying readingsfrom the mass flow sensor on an LCD (not shown) on the front panel 38 c.Preferably, each of the foregoing components are contained within thesame housing 10 a.

In operation, the nitrogen nebulizing gas is preferably regulated from45-80 psig with 99.9% purity or better. A stable gas flowrate andpressure are necessary for reproducible results. The gas is preferablyfree of contaminants, such as oil, water, particulate or any othernon-volatile substances. The droplet size in the nebulizedchromatography effluent may be regulated by varying the flow rate ofeither the chromatography effluent and/or the nebulizing gas. The lowerthe flow rate of the chromatography effluent, the less gas and heatnecessary for nebulization and subsequent evaporation.

FIGS. 2, 3 and 4 and illustrate the construction of the nebulizer 14 andthe drift tube assembly 18 in more detail. The drift tube assembly 18comprises four phenolic cover plates 40 surrounding insulation 42. Theinsulation 42 is preferably ½ inch polyimide available from McMASTERCARR, as part no. 446K121. The insulation 42, in turn, surrounds surfaceheaters 44. The surface heaters are thermoelectric heating elementspreferably of silicon pad style available from WATLOW, as part no.F030050C7. The surface heaters are preferably mounted by an appropriateadhesive on heating tube 46. Heating tube 46 is preferably made fromaluminum or other appropriate thermally conductive material. A drifttube 47 is telescopically positioned by a slip fit within heating tube46. The drift tube 47, which surrounds channel 11, is preferablyconstructed from stainless steel. A thermal fuse 43 (shown in phantom)is preferably provided for turning the heat off if temperature exceeds apre-determined limit. The fuse is in electrical communication with thepower source for the silicon heaters 44. Also preferably included istemperature sensor 54 for monitoring the temperature in the drift tubeassembly 18. The sensor is in electrical connection with logic board.

With reference to FIGS. 3, 3 a and 3 b, the nebulizer 14 comprises anebulizer tee nozzle 101, a nebulizer body 102, nebulizer adaptor 103,and tip piece 104. The nebulizer body 102 is attached to tee nozzle 101by bolts 105. Preferably, six bolts 105 are provided (see FIG. 3a). Theadaptor 103 is removably secured to the drift tube assembly 18 (see FIG.4). The tee nozzle 101, nebulizer body 102 and nebulizer adaptor 103 arepreferably made from stainless steel. O-rings 109, 109 a and 109 b areprovided to give a liquid and air tight seal between the tee nozzle 101,nebulizer body 102, and nebulizer adaptor 103. O-rings 109, 109 a and109 b are VITON O-rings available from DUPONT. Bottom seal 104 cprovides a gas and liquid seal between body 102 and adapter 103. Bottomseal 104 c is preferably a teflon/SS seal available from VERI-SEAL aspart no. W10-MH-P-012-X-1. The tip piece 104 is removably secured tonebulizer body 102 by threads 104 a and complementary threads 104 b.Openings 104 d are provided in tip 104 for receiving a two-prongedadjustment tool which assists in adjusting tip 104 relative to nebulizerbody 102.

A chromatography effluent port 108 is provided for introducing andflowing the chromatography effluent into the nebulizer 14 and tonebulizer needle 112. Chromatography effluent port is in fluidcommunication with connector 12 (see FIG. 2) by a standard nut andferrule connection. A back pressure channel 106 is also provided whichis in fluid communication with liquid back pressure relief valve 34 (seeFIG. 1a). A liquid pressure sensor channel 106 a (see FIG. 3a) is alsoprovided which is in fluid communication with liquid pressure sensor 33(see FIG. 1a). If chromatography effluent back pressure in the nozzleexceeds a pre-determined limit, the liquid back pressure relief valvediverts chromatography effluent flow away from the nebulizer throughliquid back pressure relief valve 34 to waste. Channels 106 and 106 aare in fluid connection with, liquid back pressure relief valve andliquid pressure sensor, respectively, by stainless steel tubingconnected to channels 106 and 106 a by standard nut and ferruleconnections available from ALLTECH as part nos. 206085 (nut) 286075(ferrule).

The nebulizer needle 112 comprises two concentrically positioned needleswhich have been silver soldered to each other. Preferably, there is novoid between the concentrically positioned needles. The nebulizer needle112 has a longitudinal channel through which the chromatography effluentis flowed. The nebulizer needle 112 is preferably constructed fromstainless steel. The nebulizer needle 112 is maintained in fluidconnection with chromatography effluent port 108 by nut 116. Nut 116 hasa longitudinal bore in which nebulizer needle 112 sits. Preferably, theinternal diameter of the nebulizer needle is between about 0.007 toabout 0.012 of an inch.

The nebulizer body further comprises set screws 112 a for centering thenebulizer needle 112. It is important to center the nebulizer needle sothat the chromatography effluent exiting the needle is substantially inthe concentric center of the nebulizing gas. The pressurized nebulizinggas is introduced into the nebulizer through nebulizing gas port 118 innebulizer adaptor 103. The nebulizer gas flows into channel 105 a formedbetween nebulizer adaptor 103 and shoulders 102 a and 102 b of nebulizerbody 102. A small opening 105 b is formed in the nebulizer body 102generally in the plane of the set screws 112 a (see FIG. 3b). Thepressurized nebulizing gas flowing through opening 105 b is forced intonebulizing gas chamber 130. Because seal 109 a provides a gas tightseal, the pressurized nebulizing gas is forced inside tip 104 intonebulizing zone 132. The nebulizing gas strikes the chromatographyeffluent exiting the nebulizer needle 112 in the nebulizing zone 132.The nebulizing gas breaks-up the chromatography effluent to form anaerosol of droplets. The chromatography effluent aerosol is then flowedto the drift tube assembly 18 for the evaporation step as discussedabove. In addition to set screws 112 for centering the nebulizer needle,nebulization (e.g. droplet particle size) may also be varied byadjusting tip 104.

FIGS. 3 and 4 illustrates the fluid connection between the nebulizer 14and drift tube assembly 18. The nebulizer 14 is removably secured to thedrift tube assembly 18 by operation of nebulizer bracket 16, screws 16a, nebulizer adaptor 103 and drift tube cap 118 a. The nebulizer adaptor103 has an L-shaped channel 119 for receiving the drift tube assembly18. In particular, a terminal end 18 b of the tube 47 of the drift tubeassembly 18 is positioned in void 119 a of channel 119. A clip 109 e isattached to the terminal end of tube 47 to provide a shoulder forresting upon the drift tube cap 118 a. An O-ring 109 d provides a liquidand gas tight seal at the junction of the terminal end of tube 47 withnebulizer adaptor 103. The drift tube assembly 18 further has a drifttube cap 18 a which fits into channel 119 of the nebulizer adaptor 103.Screws 119 e are provided to removably secure the drift tube cap 18 a tothe nebulizer adaptor 103. Finally, nebulizer bracket 16 has channels 16b for receiving screws 16 a. Shoulder 16 c of the nebulizer bracket 16abuts against shoulder 14 a of nebulizer 14. Shoulder 14 b of nebulizer14 abuts against surface 103 a of nebulizer adaptor 103. Thus, whenscrews 16 a are inserted through channel 16 b and into channels 103 bformed in nebulizer adaptor 103 (not shown in FIG. 3), the nebulizer isremovably secured in fluid communication with the drift tube assembly18.

As those skilled in the art will recognize, nonvolatile impurities inthe mobile phase or nebulizing gas will be detected thereby producingbaseline “noise.” By using the highest quality gas, solvents andvolatile buffers which are preferably filtered, the baseline noise willbe reduced. Baseline noise will also result from the mobile phase notbeing completely evaporated. Also, the sample may be volatilized if thedrift tube temperature is too high or the sample is too volatile. Thetemperature in the heated drift tube 18 and the flowrate for thenebulizing gas are dictated by the volatility and flow rate of themobile phase. At a mobile phase flowrate of 1 mL/min., the followingdrift tube temperatures (in ° C.) and nebulizing gas flowrates (inStandard Liters Per Minute (SLPM)) are recommended: acetone (45 C,1.50); acetonitrile (70 C, 1.70); heptane (50 C, 1.60); hexane (60 C,1.60); isopropyl alcohol (80 C, 2.20); methanol (70 C, 1.65); methylenechloride (75 C, 2.00); water (115 C, 3.20); methanol:water (90:10) (70C, 2.00); and acetonitrile:water (75:25) (90 C, 2.00).

When calculating the starting temperature and nebulizing gas flowratefor mixed mobile phases, the above values in the same ratio as themobile phase solvents are to each other should be used. Thus, if runninga binary mobile phase of 60% methanol and 40% water, the temperaturewould be (0.6)(70)+(0.4)(115)=88 C. The nebulizing gas flowrate would be(0.6)(1.65)+(0.4)(3.20)=2.27 SLPM. The above recommended temperature andnebulizing gas flowrates should be adjusted if the mobile phaseflowrates are changed from 1 mL/min. Lower mobile phase flowratesgenerally require lower nebulizing gas flowrates and temperature. On theother hand, higher mobile phase flowrates may require higher nebulizinggas flowrates and temperature.

Finally, with respect to solvents not discussed above, suitable startingdrift tube temperatures and nebulizing gas flowrates may be estimated asfollows. Obtain from an appropriate reference the solvent's boilingpoint and vapor pressure. Use the temperature and gas flowrate of thesolvent listed above that most closely matches the boiling point andvapor pressure of the solvent of interest.

Of course, some experimentation may be necessary to obtain the optimumgas flowrate, mobile phase flowrate and temperature for any particularanalysis. The nebulizing gas flow rate determines the mobile phasedroplet size. Higher flowrates produce smaller droplet sizes whichenhance vaporation. On the other hand, smaller droplets produce smallersample particles which scatter less light and produce smaller signalsfor detection. Generally, the optimal nebulizing gas flowrate is thelowest flowrate that will produce the largest peaks with an acceptable,low noise baseline. This can be determined by finding the signal tonoise ratio of various flowrates. By plotting the signal to noise ratiovs. peak area and/or the gas flowrate vs. peak area, the optimal gasflowrate may be determined.

With respect to the flowrate of the mobile phase, higher flowratesrequire higher gas flowrates and higher temperatures. It is thereforepreferable to use the lowest mobile phase flowrate possible. Thetemperature selection depends on mobile phase volatility, and flowrate,and nebulizing gas flowrate. Aqueous solvents require highertemperatures than organic solvents. Lower nebulizing gas flowratesproduce larger droplets and, therefore, require higher temperatures forevaporation. Preferably, the lowest temperature that will produce anacceptable, low noise baseline should be used. When working withtemperature sensitive samples that are volatile at the temperaturenecessary to evaporate the mobile phase, the drift tube temperature maybe decreased by increasing the nebulizing gas flowrate. However, becausesmaller droplets are produced, the increased gas flowrate will decreasedetection sensitivity of the sample.

When it is desired to convert from the single flow design as previouslydescribed to the split-flow design, when, for example, aqueous mobilephases and semi-volatile samples are present, this conversion may bequickly and easily accomplished by using the low temperature adaptor ofthe present invention. To accomplish the conversion, the nebulizerbracket 16 and nebulizer 14 are removed from the nebulizer adaptor 103by removing screws 16 a and manually removing these pieces (see FIG. 4).The low temperature adaptor is inserted in place of the nebulizer 14 asdescribed below with reference to FIG. 10. However, before discussingthe manner in which the low temperature adapter is connected to theevaporative light scattering detection device, an overview of the lowtemperature adapter construction is provided.

Isometric views of the low temperature adaptor according to oneembodiment of the invention are illustrated in FIGS. 5-6. The lowtemperature adaptor 200 has a nebulizer 14. The nebulizer 14 is the sameas the nebulizer previously described. The nebulizer 14 may either bethe nebulizer 14 removed from the evaporative light scattering device 10before attaching the low temperature adaptor, or it may be a secondnebulizer. The nebulizer 14 is connected to a nebulization chamber 208by nebulizer union 206. Details concerning the manner of attachment ofthe nebulizer 14 to the nebulizer union 206 are discussed below withreference to FIGS. 8a and 8 b. The nebulizer union 206 is preferablymade from stainless steel and is friction fit to the nebulizationchamber 208 by O-ring 246. The nebulization chamber 208 has a sink trapdrain 212. A tapered connector 216 attaches the nebulization chamber 208to coil 218. The connector 216 is preferably made from stainless steeland is welded to the chamber 208 and coil 218. The chamber 208 and coil218 are also preferably made from stainless steel. The coil 218 ispreferably ½ inch or 1 inch in diameter. The foregoing pieces arepreferably contained in housing 222.

A gas filter in-line 226 is preferably provided for supplying the sweepgas to the nebulizer union member 206 as described below. A backpressure line 227 is also preferably provided to flow chromatographyeffluent away from the nebulizer 14 to waste when chromatographyeffluent exceeds a pre-determined pressure limit. The coil 218 exitshousing 222 to flow the nebulized chromatography effluent to theevaporative light scattering detection device via connection tube 260described herein. Finally, the housing 222 also preferably includespower module 232, temperature controller 233, sweep gas regulator 209,back pressure regulator 235, sold state relay 236, and mass flowcontroller 248.

Sweep gas regulator 209 regulates the flowrate of the sweep gasintroduced to the nebulizer 14 from a sweep gas source (not shown).Preferably, the sweep gas is selected from the same gas as thenebulizing gas. Most preferably, the sweep gas is nitrogen. Suitabletubing (not shown) provides a gas connection between the sweep gasregulator 209 and the nebulizer union 206. Back pressure regulator 235is in fluid connection with nebulizer 14 by stainless steel. If the backpressure exceeds a pre-determined level, the back pressure regulatordiverts chromatography effluent flow from nebulizer 14 through backpressure regulator 235 to back pressure waste line 235 a (not shown inits entirety). Back pressure waste line 235 a exits the low temperatureadapter housing 222 and flows the chromatography effluent to waste. Apreferred back pressure regulator is a diaphragm back pressure regulatoravailable from the assignee of this application, ALLTECH ASSOCIATES,INC. The solid state relay 236 is in electrical connection with the heattape 228 affixed to nebulization chamber 208 and coil 218 andtemperature controller 233. The solid state relay turns the heat tape“on” and “off” in response to the temperature controller 233. Apreferred temperature controller is a PID action, TC input availablefrom WATLOW, as part no. 965A3CA000BR. A preferred solid state relay isavailable from NEWARK, as part no. 27F329. The mass flow controller 248controls the flow of nebulizing gas to the nebulizer 14. The mass flowcontroller 248 is in gas communication with the nebulizer 14 and thenebulizing gas source (not shown) by suitable tubing, as for exampleTEFLON tubing. A preferred mass flow controller is as previouslydescribed with respect to the evaporative light scattering device 10.

The low temperature adapter preferably uses one gas source for both thenebulizing gas and the sweep gas. Preferably, the gas (which ispreferably nitrogen) enters the low temperature adapter housing 222 at“gas in” line 239. After entering the low temperature adapter, the gasis split into two gas streams. The first stream, the sweep gas is flowedto a sweep gas filter (not shown) for removing impurities from the sweepgas. The sweep gas is then flowed from the filter to the sweep gasregulator 209 and nebulizer 14, respectively. The second stream, thenebulizing gas, is flowed out of the low temperature adapter housing 222via “ELSD out” line 239 a. The nebulizing gas is flowed into theevaporative light scattering device 10 where it is filtered bynebulizing gas filter 30 in the scattering device 10. After filtering toremove impurities, the nebulizing gas is flowed through mass flow sensor36 in the scattering device 10 and back to the low temperature adapterthrough “ELSD in” line 239 b to mass flow controller 248 andnebulization chamber 208, respectively.

With reference to FIG. 7, heat tape 228 is preferably wrapped around thenebulization chamber 208 and the coil 218 at predetermined intervals. Byvarying the amount of heat tape on the nebulization chamber 208 and thecoil 218, a heat application gradient may be established. Preferably,heat is applied at a higher rate at the nebulization chamber 208 than atthe coil 218. The majority of evaporation takes place in thenebulization chamber, and the surface area per unit length in thenebulization chamber 208 is greater than in the coil 218. If there isnot enough heat in the nebulization chamber, a greater percentage of themobile phase will pass through the chamber and ultimately to the opticalcell for detection. This will result in an unstable baseline and hinderdetection of the sample. On the other hand, if there is too much heatapplied at the coil 218, there is a risk of evaporating sample whichwill reduce the amount of sample detected. By asymmetrically applyingthe heat tape at the nebulization chamber 208, and the coil 218, moreheat may be delivered to the nebulization chamber 208 than to the coil218 using the same heat source. When ½ inch coil is used, it ispreferred to leave ½ to ¾ inch spacing between the heat tape. When 1inch coil is used, it is preferred to leave ⅛ inch spacing between theheat tape. Most preferably, the amount of heat tape per surface area isgreater in the nebulization chamber 208 than in coil 218. Mostpreferably, the ratio of applied heat per unit surface area in thenebulization chamber 208 to the coil 218 is about 1:1 to about 3:1 andmost preferably about 1.7:1. The heat tape 228 is preferably cut from ½inch width H-series heat tape. Preferred heat tape is available fromCLAYBORN, under part no. J-16-4. The heat tape is in electricalconnection with power module 232 in the low temperature adaptor housing222 (see FIG. 5).

FIGS. 8a and 8 b further illustrates the connection between thenebulizer 14 and the nebulizer union 206 of the low temperature adaptor.The nebulizer consists of tee nozzle 101, nebulizer body 102 and tip 104and is as previously described with reference to FIGS. 3, 3 a, and 3 b.Chromatography effluent is delivered to tee nozzle 101 by the same meanspreviously described with respect to connector 12 and chromatographyeffluent line 12 a. The union 206 has a sweep gas port 240 and sweep gaschannel 241, which are in gas communication with nebulization chamber208. Port 240 and channel 241 are for introducing sweep gas to thenebulization chamber 208. Port 240 is in gas communication with sweepgas regulator 209 (see FIGS. 5 and 6). The sweep gas is preferablyintroduced into the same plane as the tip of the nebulizer needle andparallel to the nebulizing gas. The purpose of the sweep gas is toassist in evaporating the mobile phase and to provide a way ofcontrolling the evaporation rate without affecting nebulization andparticle size distribution in the mobile phase. The nebulizer union 206further includes nebulizing gas port 242 which introduces nebulizing gasto the nebulizer as previously described with respect to FIGS. 3, 3 a,and 3 b. Nebulizing gas flow rate is controlled by any suitable gasvalve or valving arrangement. Preferably, the nebulizing gas isdelivered from the same nebulizing gas source as used by the evaporativelight scattering detection device. Seal 246 provides a gas tight sealbetween the inside wall 251 of nebulization chamber 208 and nebulizerunion 206. This seal is preferably EPDM rubber seal available fromMcMASTER CARR, Chicago, Ill., as part no. 9557K19. Nebulizer 14 isremovably attached to union 206 by screw 505.

FIG. 9 illustrates the flow path and method of operation when the lowtemperature adaptor is used in conjunction with the evaporative lightscattering device. The chromatography effluent is flowed through anebulizer 14 and nebulizer union 206. The nebulized chromatographyeffluent is flowed to nebulization chamber 208 where the larger dropletsof the mobile phase collide with the inside walls of the chamber 208 andare flowed to waste via sink trap 212. By removing the larger particlesin the chromatography effluent, a smaller droplet size distribution iscreated and the temperature required to evaporate the remaining dropletsof the mobile phase is reduced. Hence the name “low temperatureadaptor.” To avoid widely fluctuating temperatures that may adverselyaffect reproducibility, the temperature of the nebulization chamber 208is precisely controlled with the heat tape 228. Evaporation of themobile phase begins in the nebulization chamber 208. The “split”nebulized chromatography effluent is then flowed through heated coil 218where evaporation of the mobile phase continues. Because of the smallerparticle size distribution in the mobile phase, the evaporationtemperatures used in the coil 218 are lower than if the chromatographyeffluent was not first split in the nebulization chamber 208. Byoperating at a lower evaporation temperatures, the low temperatureadaptor improves the sensitivity of the system to semi-volatile samples.The sample, which has not been evaporated, is then flowed throughconnection tube 260 and drift tube assembly 18, respectively, todetection zone 19.

FIG. 10 illustrates the interface between the low temperature adaptorand the evaporative light scattering device. The coil 218 is secured tothe bottom of housing 222 for the low temperature adaptor by coilconnection plate 250. The connection plate 250 is welded to the coil 218and is removably secured to the housing 222 by hex head nuts 251 andsocket head cap screws 253. A channel 256 extends along a vertical axisthrough connection plate 250. A shoulder 257 is positioned in channel256. A lower end of coil 218 abuts against one side 257 a of shoulder257. A connection tube 260 is inserted into channel 256 until it abutsagainst the opposite side 257 b of shoulder 257. An O-ring 261 isprovided to form a gas and liquid tight seal between connection tube 260and the inside wall of channel 256 of connection plate 250. O-ring 261is preferably EPDM rubber such as McMASTER CARR part no. 9557K2-018.

The connection tube 260 (which is preferably made from stainless steel)is in fluid communication with tube 47 of drift tube assembly 18 of theevaporative light scattering device 10. In particular, the housing forthe evaporative light scattering device 10 has a recess 10 a. The recess10 a is exposed by removing a removable cover cap (not shown) from thehousing of evaporative light scattering device 10. The removable covercap is configured to cover the recess 10 a when the system is in thesingle flow configuration and, therefore, the low temperature adaptor isnot on-line. Sitting in recess 10 a is the drift tube assembly 18.Attached to the drift tube assembly 18 is drift tube cap 118 a. Thenebulizer adaptor 103 is removably secured to the drift tube cap 118 aby screws 119 e as previously described. Nebulizer adaptor 103 has screwchannels 103 b for receiving screws 16 a. Connection tube plate 266 is,therefore, removably secured to nebulizer adaptor 103 by screws 16 a.Connection tube plate 266 has a bore for receiving connection tube 260.Connection tube seal 280 provides a gas and liquid tight seal betweenthe connection tube 260 and connection tube plate 266. Connection plateseal 282 provides a gas and liquid tight seal between connection tubeplate 266 and nebulizer adaptor 103. Seal 280 is the same type as seal260 and seal 282 is the same type as seal 246. As can be ascertainedfrom comparing FIGS. 4 and 10, the low temperature adaptor may bequickly and easily attached to the evaporative light scattering device10 by removing the nebulizer bracket 16 and nebulizer 14, and theninserting connection tube 260 through nebulizer adaptor 103, applyingconnection tube and connection plate seals 280 and 282, respectively,and securing screws 16 a. By following these simple steps and re-routingnebulizing gas flow and the chromatography effluent flow, an evaporativelight scattering detection device 10 may be quickly and easily convertedbetween the single flow and split flow designs.

The low temperature adaptor is intended to operate at low temperaturesand low nebulizing gas flow rates. The low temperature adaptor will bepreferably operated in a temperature range of ambient to 100 C in 1Cincrements. The nebulizing gas is preferably nitrogen with a pressurerange of about 45 to about 80 psig and a flow rate of (0-5 SLPM). Themobile phase flowrate is preferably about 0.1 to about 5.0 mL/min. Thetemperature controller is preferably a microprocessor based PIDtemperature controller. The mobile phase flow path is preferably madefrom stainless steel. The gas flowpath is preferably made from TEFLONtubing. In general, an operating temperature of about 40 C and anebulizing gas flow rate of 1.75 SLPM is sufficient for mostapplications. However, to obtain maximum detector response for eachapplication, some experimentation may be necessary to determine optimumtemperature and nebulizing gas flowrate.

Temperature selection depends mainly on the volatility of the mobilephase used, but is also affected by the mobile phase flowrate. Aqueoussolvents require slightly higher temperatures than organic solvents. Atemperature of about 40 C is sufficient to evaporate mobile phasesconsisting of 100% water at flowrates up to 2.0 mL/min. and, therefore,is a good starting point. Mobile phases containing a large portion oforganics may require temperatures as low as ambient (25 C). The lowtemperature adaptor is preferably not used with such mobile phasesunless semi-volatile samples are involved. Most preferably, the lowesttemperature that produces an acceptable, low noise baseline should beused for most applications. It should also be noted that the lowtemperature adaptor and the evaporative light scattering device shouldbe operated at the same temperature.

The nebulizing flowrate selection will also depend on mobile phasevolatility and mobile phase flowrate. Preferably, nebulizing gasflowrates will be under 2.0 SLPM, unless extremely high mobile phaseflowrates are used. For low mobile phase flowrates or highly organicmobile phases, nebulizing gas flowrates may be as low as 1.0 SLPM. Mostpreferably, the lowest gas flowrate that produces an acceptable, lownoise baseline should be used.

In general, when using non-volatile samples and organic mobile phases,the single flow evaporative light scattering device may be used.However, when switching to semi-volatile sample types, aqueous mobilephases and/or higher mobile phase flowrates, the evaporative lightscattering device of the present invention may be quickly and easilyconverted to a split flow design as described herein.

Following are examples demonstrating how to use the invention disclosedherein. With respect to the examples using the low temperature adapter,unless otherwise stated, nitrogen sweep gas was used at 2 SLPM. In theexamples, heat tape was affixed to the coil and nebulization chambersuch that the amount of heat tape on the nebulization chamber per unitservice area was 1.7 times greater than that affixed to the coil.

EXAMPLES Example 1

FIGS. 11 and 12 demonstrate the improved baseline stability obtainedwhen using the low temperature adaptor (LTA) in combination with theevaporative light scattering detection (ELSD) device with a highlyaqueous mobile phase. When highly aqueous mobile phases are used for theseparation, higher drift tube temperatures are needed. The LTA permitseffective detection of lower evaporation temperatures. FIG. 11 is achromatogram of the ELSD alone. FIG. 12 is a chromatogram of theLTA/ELSD combination. The separation column was an Econosphere C18, 3μm, 30×4.6 mm; the mobile phase was methanol: water: acetic acid(38:62:1); the flowrate 1.5 mL/min; sample size was 0.2 mg/mL caffeine,0.8 mg/mL aspirin. With respect to FIG. 11, drift tube temp. 95° C., 20μL loop, nebulizing nitrogen flow rate 3.70 SLPM. With respect to FIG.12, drift tube temp. 50° C.; nitrogen flow 2.5 SLPM; nebulizer chambertemp. 38° C.; coil temp. 60° C.; nitrogen sweep gas flow 3 SLPM.

Example 2

The ELSD alone is preferred for non-volatile samples or organic mobilephases. The ELSD alone is preferred when analyzing non-volatilecompounds or when using organic mobile phases. FIGS. 13 and 14demonstrate this. Organic mobile phases evaporate easily, reducingoperating temperatures so that sample integrity is preserved. When usingthe ELSD alone, all of the sample enters the optical cell, maximizingresponse. With respect to FIGS. 13 and 14, separation was by HPLC.Column was Adsorbosphere C18, 5 μm, 250×4.6 mm. Mobile phase wasmethanol: acetonitrile (97:3); the sample size was 20 μL injection loop,mobile phase flowrate 1.0 mL/min. With respect to FIG. 13, the drifttube temp. was 70° C. and nebulizing nitrogen flow was 2.00 SLPM.

Example 3

The ELS/LTA combination is preferred with semi-volatile samples. The LTAlowers the ELSD's operating temperature, eliminating semi-volatilesample loss to evaporation. This preserves sample integrity andmaximizes response. This is demonstrated by FIGS. 15 and 16. FIG. 15 isa chromatogram of the ELSD/LTA combination and FIG. 16 is achromatograph of the ELSD alone. Separation was by HPLC. Column wasAlltima C18-LL, 5 μm, 250×2.1 mm. Mobile phase was a gradient of water:acetonitrile (Time (min.): % acetonitrile: 0:77, 10:80, 15:80, 20:95);flowrate 0.4 mL/min; sample size 20 μL loop. With respect to FIG. 15,drift tube and LTA temp. 30° C.; and nebulizing nitrogen flow 1.75 SLPM.With respect to FIG. 16, drift tube temp. 65° C., and nebulizingnitrogen flow 2.0 SLPM.

Example 4

The ELSD delivers a stable baseline and excellent sensitivity for asimple sugar separation. Because carbohydrates are non-volatile and themobile phase is mostly organic, the ELSD alone is preferred. FIG. 17demonstrates this. The separation was by HPLC. Column was Absorbosphere,NH₂ 250 mm×4.6 mm, sample size was 1 mg sugar standards/ml, mobilephase. Acetonitrile: water (85:15), flowrate 1.5 mL/min., drift tubetemp. 90° C., nebulizing nitrogen flow 2.20 SLPM.

Example 5

The ELSD with the LTA detects corn syrup oligomers under gradientconditions. The ELSD/LTA combination is preferred for this applicationbecause of the high flowrate and highly aqueous mobile phase. TheELSD/LTA combination maintains a stable baseline during the gradient.

Example 6

Dimethicone analysis using the ELSD combined with non-aqueous reversedphase gradient elution achieves good resolution and detectionsensitivity. Because dimethicone is a large non-volatile molecule andthe mobile phase is 100% organic, the ELSD alone is preferred.

Example 7

The LTA maximizes sensitivity in the analysis of PEG 200. The LTAreduces the ELSD's operating temperature and enhances the detectionsensitivity of this small, semi-volatile compound. Thus, FIG. 18 is achromatogram demonstrating the preferred results with the ELSD/LTAcombination. Separation by HPLC. Column was Econophere C8 5 micron250×4.6 mm, mobile phase water: methanol gradient (Time (min): %methanol: 0:15, 25:40, 35:40), flowrate 1.0 mL/min., sample size 1mg/mL, drift tube and LTA temp. 30° C., nebulizing nitrogen flow 1.75SLPM, and sweep gas 2.5 SLPM.

Example 8

The ELSD alone is preferred for samples such as phospholipids. Thisconfiguration is ideal for normal phase applications. FIG. 19demonstrates this. The separation was by HPLC. Column was AllsphereSilica, 3 μm, 100×4.6 mm, mobile phase gradient of IPA: Hexane: Water(Time (min.): % IPA: % Hexane: % Water: 0:58:40:2; 7:52:40:8; 15:52:40:8, flowrate 1.25 μL/min., column temp. ambient; drift tube temp. 65°C., nebulizing nitrogen flow 2.0 SLPM.

Example 9

The LTA enhances detector sensitivity in the analysis of underivatizedlow-chain fatty acids. The LTA substantially lowers the ELSD's operatingtemperature, preventing sample loss to evaporation. A chromatogram usingthe preferred combination of the ELSD/LTA is shown in FIG. 19. Theseparation was by HPLC. Column was Alltima C18, LL, 5 mm (250×2.1 mm),sample size 20 μL injection loop, mobile phase, gradient water:acetonitrile (time (min.): % of acetonitrile): 0:77, 5:80, 10:80, 20:95;flowrate 0.4 ml/min., drift tube and LTA temp. 30° C., and nebulizingnitrogen flow 1.25 SLPM.

Example 10

Large macrolides are not subject to sample evaporation, therefore theELSD alone is preferred.

Example 11

Assessing lead drug purity is preferred using the ELSD/LTA combinationcompared to UV because the ELSD's signal closely reflects the sample'smass balance. The LTA accepts high flowrates and operates at lowtemperatures for the extreme gradient conditions used duringhigh-throughput screening. This is demonstrated by FIGS. 23 and 24. FIG.23 is a chromatogram from using UV detection. FIG. 21 is a chromatogramgenerated by the preferred ELSD/LTA combination. The separation was byHPLC. Column was Alltima C18, 5 mm, 50×2.1 mm, mobile phase gradient ofwater (0.1% formic acid): acetonitrile (0.1% formic acid) (time (min): %acetonitrile): 0:5, 10:95, 11:95; flowrate 0.5 mL/min., column temp. 40°C. With respect to FIG. 23, detection by UV at 220 nm. With respect toFIG. 24, LTA drift tube and LTA temp. 30° C., and nebulizing nitrogenflow 1.75 SLPM.

We claim:
 1. A method for quickly converting from a single-flowconfiguration to a split-flow configuration of evaporative lightscattering detection thereby providing increased ability to detectdifferent sample types in different mobile phases, the methodcomprising: providing an evaporative light scattering detection devicein the single-flow configuration comprising a nebulizer in fluidcommunication with one end of a drift tube, a light source and adetector; and converting to a split-flow configuration by removing thenebulizer and connecting a low temperature adaptor such that the lowtemperature adaptor is in fluid communication with the drift tube, thelow temperature adapter comprising a low temperature adapter nebulizer,a nebulization chamber in fluid communication with the low temperatureadapter nebulizer and the drift tube, wherein in the nebulizationchamber at least a portion of a mobile phase carrying sample componentsis evaporated and a portion of the mobile phase is not evaporated, thenebulization chamber further comprising a sink trap in fluidcommunication with the nebulization chamber for removing mobile phasethat is not evaporated in the nebulization chamber.
 2. The system ofclaim 1 wherein the low temperature adapter has a sweep gas channel forintroducing sweep gas into the nebulization chamber.
 3. The system ofclaim 1 wherein heat tape is affixed to the nebulization chamber of thelow temperature adaptor at pre-determined intervals.
 4. The method ofclaim 1 wherein the nebulizer and the low temperature adapter nebulizerare the same.
 5. The system of claim 4 wherein heat tape is affixed tothe coil of the low temperature adaptor at pre-determined intervals. 6.The method of claim 1 wherein the nebulization chamber of the lowtemperature adapter is in fluid communication with the drift tube via aconnection tube.
 7. The method of claim 6 wherein the connection tubeand the nebulization chamber are in fluid communication via a coil. 8.The method of claim 1 comprising the further step of converting back tothe single-flow configuration by removing the low temperature adapterand attaching a nebulizer such that it is in fluid communication withthe drift tube.
 9. The method of claim 8 wherein the nebulizer and thelow temperature adapter nebulizer are the same.